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Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli

A technology for recombining Escherichia coli and phospholipase, which is applied in the fields of genetic engineering and microbial fermentation, can solve the problems of difficult large-scale production, limited extraction, high price, etc., and achieves the effects of short production cycle, easy process amplification and low cost.

Active Publication Date: 2013-05-01
JIANGNAN UNIV +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The traditional source of phospholipase A1 is animal pancreatic juice, but the extraction amount of this source is limited and expensive, and it has been gradually eliminated in recent years. Most of the phospholipase A1 is bound to the cell membrane, and it is difficult to carry out large-scale production. Therefore, it is important to study the cloning and expression of the phospholipase A1 gene in engineering bacteria and strive to improve its production. The main research directions in recent years
Phospholipase A produced by microorganisms at home and abroad 1 It started late, and the yield is generally low: in 1988, Michael Givskova et al. extracted phospholipase A from Serratia liquefaction 1 The gene was cloned and expressed in Escherichia coli, and the enzyme activity was less than 1U / ml (Givskov M, Molin S.Secretion of Serratia liquefaciens phospholipase from Eseherichia.coli[J].Mol.Microbiol, 1993(8):229-42.) In 2000, M.Hartmann et al. used random chemical mutation, single-cell fusion and gene hybridization techniques to screen out 4 mutant strains of Tetrahymena thermophila, with the highest enzyme activity reaching 35.2±2.60U / ml (Hartmann M, et al. .Screening for and charaeterization of phospholipase A 1 hypersecretory mutants of Tetrahymena thermophila[J].Appl.Microbiol.Biotechnol, 2000(54):390-396); Phospholipase A 1 The strain xjF1, after preliminary optimization, has an enzyme activity of up to 17.5U / ml (Fu Jianhong, Tang Huigui, Yao Bin, etc. A strain producing low-temperature alkaline phospholipase A 1 Screening of cold-resistant bacteria and preliminary study on fermentation conditions. Industrial Microbiology 2008.38: 12-16.)

Method used

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  • Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli
  • Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli

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Embodiment 1

[0029] Example 1 Phospholipase A 1 gene cloning

[0030] Design primers according to the gene sequence of NCBI GenBank: X66505.1, where the upstream primer is: 5′-CG GAATTC AGTATGTCTTTGAGTTTTACCTCTGC-3' (the underline indicates the EcoR Ⅰ restriction site), the downstream primer is: 5'-CC AAGCTT CGTTACTGCTGTCCGTATTGC-3' (the underline indicates the HindⅢ restriction site).

[0031] Phospholipase A was cloned by using the PCR method (primers as above) using Serratia liquefaction genomic DNA with the preservation number CICC No. 21538 as a template 1 The gene pla was connected with the vector pMD18T-simple to obtain the cloning vector pMD-pla. The cloning vector was transformed into Escherichia coli competent cell E.coli JM109, and positive clones were selected and verified by sequencing. Its base sequence is shown in SEQ ID NO: 1 showed that the results showed that phospholipase A 1 Gene cloning was successful. Sequence analysis showed that the gene sequence was identical...

Embodiment 2

[0032] The construction of embodiment 2 recombinant vector pET-pla

[0033] The recombinant vector pMD-pla and the expression vector pET-28a (+) were double digested with EcoR Ⅰ and Hind Ⅲ. The enzyme digestion reaction system was 50 μL: vector 20 μL, enzyme reaction buffer 5 μL, EcoR Ⅰ 2.5 μL, Hind Ⅲ 2.5 μL, supplemented Double-distilled deionized water to 50 μL, reacted at 37°C for 2 hours. Recover the digested target gene and carrier DNA, and connect them with T4 DNA ligase, ligation reaction system 10 μL: target gene 2 μL, carrier DNA 1 μL, 10×T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, double distilled deionized water 5 μL , reacted at 16°C for 12 hours.

[0034] Transform the ligation product into Escherichia coli competent cells E.coli JM109, the transformation method is as follows:

[0035] (1) Take 100 μL of E.coli JM109 and place it in a sterile 1.5ml EP tube;

[0036] (2) Add the above ligation product and mix gently;

[0037] (3) Put the above EP tube in a metal...

Embodiment 3

[0042] Example 3 Construction of recombinant Escherichia coli BL21(DE3) / pET-pla CCTCC M 2012423

[0043] Transform the constructed recombinant vector pET-pla into Escherichia coli BL21 (DE3), the transformation method is as follows:

[0044] (1) Take 100 μL of E.coli BL21 (DE3) and place it in a sterile 1.5ml EP tube;

[0045] (2) Add the above recombinant plasmid pET-pla and mix gently;

[0046] (3) Put the above EP tube in a metal bath at 42°C, accurately time it for 90 seconds, and do not shake the EP tube;

[0047] (4) Transfer the EP tube to an ice bath and cool for 5-10 minutes;

[0048] (5) Add 800 μL of sterile SOC medium to each tube, and place in a 37°C incubator to recover for 1 hour;

[0049] (6) Centrifuge at 8000r / min for 2min, suck off 800μL of supernatant medium, gently blow and suck the remaining medium and cells with a pipette gun, and transfer to LB solid containing final concentration of 50μg / ml kanamycin Plate, spread evenly and incubate in a 37°C incu...

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Abstract

The invention provides a recombinant Escherichia coli, and a method for preparing phospholipase A1 through using the recombinant Escherichia coli. A gene-recombinant Escherichia coli BL21(DE3) / pET-pla CCTCC M 2012423 with the phospholipase A1 from liquefied Serratia liquefaciens CICC No.21538 is successfully constructed in the invention, and undergoes liquid fermentation to prepare hemolytic phospholipase A1, and the preparation method of the hemolytic phospholipase A1 comprises the steps of inclined plane culture, seed culture, autonomous induction culture (comprising permeable substance addition), and crude enzyme liquid extraction. The method for preparing the phospholipase A1 through the recombinant Escherichia coli has the advantages of high enzyme yield, high enzyme activity, simple production technology, short production period, low cost, easy technological amplification and the like, and can be widely applied to the grease refining field, the phosphatide modification field and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and in particular relates to a strain of recombinant Escherichia coli and the preparation of phospholipase A by self-induced fermentation of the strain 1 Methods. Background technique [0002] Phospholipase A 1 (PhospholipaseA 1 , EC 3.1.1.32, referred to as PLA 1 ) belongs to hydrolase, which can specifically hydrolyze the Sn-1 acyl group of natural phospholipids to obtain Sn-2 acyl lysophospholipids and free fatty acids. Phospholipase A 1 It is an excellent surfactant and is widely used in the fields of food, medicine, feed and leather; in addition, phospholipase A 1 It can also be applied to vegetable oil degumming. Compared with traditional degumming methods, phospholipase A 1 Degumming, a new type of degumming method, has the advantages of mild reaction conditions, less by-products, energy saving and environmental protection, and has been widely p...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/18C12R1/19C12R1/425
Inventor 张梁石贵阳延晋雷丁重阳顾正华
Owner JIANGNAN UNIV
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